Scandinavium lactucae sp. nov. Isolated from Healthy Lettuce in South Korea

Four Gram-negative, facultative anaerobic, oxidase-negative and catalase-positive strains were isolated from lettuce sample collected from test beds at the National Institute of Agricultural Sciences in Wanju, South Korea. The whole genome sequences of the strains ranged from 4,624,629 to 4,849,846 bp in size, with DNA G + C contents of 54.32 to 54.56 mol%. Phylogenetic analyses based on 16S rRNA gene and four housekeeping (atpD, gyrB, infB, and rpoB) gene sequences showed that the four strains clustered closely together with Scandinavium type strains within the Enterobacteriaceae family. Moreover, the average nucleotide identity and digital DNA-DNA hybridization value of the proposed type strain (V105_6T) with the closely related Scandinavium type strains were in the range of 85.71–86.16% and 30.2–31.2%, respectively, which were all below the species delineation threshold values. The major cellular fatty acid of V105_6T was C16:0. Growth was observed at 7, 10 and 35 °C, and in the presence of 7% NaCl concentration. Based on phenotypic and genotypic results, strain V105_6T represents a novel species of the genus Scandinavium, for which the name Scandinavium lactucae sp. nov. is proposed. The type strain is V105_6T (= LMG 33389T = DSM 117134T). Supplementary Information The online version contains supplementary material available at 10.1007/s00284-024-03811-9.


Introduction
The Enterobacteriaceae family represents a diverse group of Gram-negative bacteria, which are facultative anaerobic, oxidase-negative and catalase-positive, and have been isolated from various environments including water, soil and the intestinal tracts of humans and animals, where they can lead to a variety of infections [1].Numerous commensal and pathogenic species within this family possess antibiotic resistance genes and harbor mobile genetic elements that can facilitate the transfer of antibiotic resistance determinants [2].For this reason, antibiotic-resistant Enterobacteriaceae are emerging as global public health threats and pose major challenges in the treatment of infections [3].
In a previous study, the novel genus Scandinavium was added to the family Enterobacteriaceae [2].The novel species within this genus, named Scandinavium (S.) goeteborgense, was isolated from a wound infection of an adult patient in Sweden and it carries a quinolone resistance gene variant (qnrB96) which exhibited high resistance to the antibiotic ciprofloxacin.This single strain was identified by sequencing of the complete 16S rRNA gene and whole genome sequencing analyses, including average nucleotide identity (ANI) and in silico DNA-DNA similarity analysis [2].In 2022, three additional species, namely Scandinavium hiltneri, Scandinavium manionii and Scandinavium Jiwon Park and Sieun Park have contributed equally to this work.
The GenBank accession number for the 16S rRNA gene sequence of strain V105_6 T (= LMG 33389 T = DSM 117134 T ) is PP412028, and that for the whole genome sequence is JAWXRD000000000.The Sequence Read Archive (SRA) accession number of the raw whole genome sequence data is SRX24351625.
tedordense were described within the Scandinavium genus [4].The type strains of S. hiltneri H11S7 T and S. manionii H17S15 T were isolated from Quercus robur rhizosphere soil in Hatchlands, Guildford, United Kingdom, while the type strain of S. tedordense TWS1a T was obtained from a bleeding lesion on a Tilia x europaea tree in Tidworth, Wiltshire, United Kingdom [4].
The global consumption of fresh produce has significantly risen, reflecting an increasing trend in dietary preferences worldwide.However, consuming it raw exposes individuals to potentially harmful bacteria, including antibiotic-resistant.With the increasing demand for fresh produce and the growing preference for raw consumption, understanding the microbial composition and antibiotic resistance profiles of lettuce is crucial for assessing public health risks and ensuring food safety [5].
In the present study, four strains obtained from lettuce exhibited high 16S rRNA gene sequence similarity to species of the Scandinavium genus.Additionally, multi locus sequence analysis (MLSA), overall genome related indices (ANI and dDDH), and whole genome-based phylogeny of these strains together with closely related type strains in this genus indicated that the isolates clearly differed from other Scandinavium species type strains and represented a novel species, for which the name S. lactucae is proposed.

Strain Isolation and Propagation
All strains were isolated from healthy lettuce (Lactucae sativa) sample in the same batch collected from test beds at the Korean National Institute of Agricultural Sciences (NAS) between 29 June and 28 July 2021 on violet red bile dextrose agar (VRBD; Merck, Darmstadt, Germany) plates [6].All strains were routinely cultured in Luria-Bertani (LB; Roth, Karlsruhe, Germany) broth at 35 °C for 18 h.For longterm storage, the isolates were stored in the same medium at −70 °C with 25% (v/v) glycerol.

Phenotypic Characterization and Cell Morphology
Colony morphology was determined on LB (Roth) agar after incubation at 35 °C for 18 h.Strains were incubated further at 7, 10, 35, and 41 °C for up to 120 h to determine the optimal temperature range for growth.To test salt tolerance, stains were inoculated in LB (Roth) broth with NaCl concentrations ranging from 1 to 10% and were incubated at 35 °C for up to 120 h.The pH tolerance of the strains was tested by adjusting the pH of tryptone soy broth (TSB, Oxoid) from 4 to 12 using 1 N hydrochloric acid (Roth) and 1 N sodium hydroxide (Roth), followed by incubation at 35 °C for 120 h.The miniaturized identification system ID 32 E (BioMerieux, Nürtingen, Germany) was used for phenotypic identification based on biochemical reactions such as enzyme activities and sugar fermentations.For this, all isolates were first sub-cultured on a LB (Roth) agar plate at 35 °C for 18 h.A single colony was picked, suspended in 0.85% NaCl with turbidity adjusted to 0.5 McFarland.Each test strip was inoculated with the suspension and incubated per the manufacturer's instructions.

Phylogenetic and Genomic Analyses
To extract total genomic DNA of the isolates, the peqGOLD bacterial DNA extraction kit (VWR, Darmstadt, Germany) was used according to the manufacturer's instructions.DNA concentration and quality were measured with a Qubit 3 fluorometer (Invitrogen, Darmstadt, Germany) and a Nan-oDrop spectrophotometer (Peqlab, Erlangen, Germany).
The almost complete 16S rRNA gene sequences of each isolate were obtained using Sanger sequencing (Microsynth, Göttingen, Germany) and analyzed with EzBioCloud [16] for identification of the isolates.A phylogenetic tree of 16S rRNA gene sequences was constructed with MEGA (v.10) [17] using maximum-likelihood method and Kimura twoparameter model [18] along with 1000 bootstrap analysis.The four housekeeping genes, ATP synthase beta subunit (atpD), DNA gyrase subunit B (gyrB), initiation translation factor 2 (infB), and RNA polymerase beta subunit (rpoB) were extracted from whole genome sequence data and multilocus sequence analysis (MLSA) was conducted with the type strains of the previously described species of the genus Scandinavium and other type strains of the family Enterobacteriaceae using the MEGA (v.10) program [17].The maximum-likelihood method and Kimura twoparameter model were applied, with bootstrap values based on 1000 replications.Additionally, a phylogenetic tree based on whole genome sequences was reconstructed with the types strains of previously described species of the genus Scandinavium, as well as selected Enterobacteriaceae type strains, using the BV-BRC server [11].For this, 100 conserved coding sequences were extracted and concatenated.A homologous group filtering and sequence alignment were performed using MAFFT pipeline which was implanted to BV-BRC, and a phylogenetic tree was generated using the RAxML pipeline with 100 bootstrap values.In order to identify all isolates precisely, average nucleotide identity (ANI) and in-silico DNA-DNA hybridization (dDDH) values were calculated using OrthoANI (v.0.93.1)[19] supported by BLAST tool [20] and the Genome-to-Genome Distance Calculator (v.3.0) with formula 2, respectively [21].A heatmap was generated using R (v. 4.3.2.) [22] to assess clonal relationship among isolates.

Cellular Fatty Acid Analysis
Analysis of the cellular fatty acid profile of the novel species candidate V105_6 T was carried out commercially by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) (Braunschweig, Germany).Briefly, cells were grown on LB agar plate at 35 °C for 18 h.The cellular fatty acids were converted into fatty acid methyl esters (FAMEs) by saponification and methylation, and extracted according to instruction's manual of the Microbial Identification System (MIDI Inc.; v. 6.1) [24].The FAMEs were separated by a GC-MS run on an Agilent GCMS-7000D system, detected by a flame ionization detector, and identified using Sherlock Microbial Identification System (TSBA; v. 6.1 library) [25].

Colony Morphology and Phenotypic Characteristics
Scandinavium sp.strain V105_6 T formed cream colonies of 1-2 mm size when incubated at 35 °C for 18 h on LB (Roth) agar.The colony shape, margin, and elevation of all strains were circular, entire, and flat, respectively.All strains were Gram-negative and exhibited growth at temperatures of 7, 10, and 35 °C, while no growth was observed at 41 °C.They showed tolerance to concentrations ranging from 1-7% NaCl, with particularly weak growth in the presence of 7% NaCl.In addition, the growth of all isolates was observed in TSB (Roth) at a pH range of 4-11, while strain V105_16 exhibited no growth at pH 4.

Phylogenetic and Genomic Analyses
The whole genome sequencing results are summarized in Table 1.The genomes of strains V105_1, V105_6 T , V105_12 and V105_16 comprised 32, 40, 42, and 45 contigs with a total length of between 4,645,037 bp (V105_12) and 4,854,890 bp (V105_1).The coding sequences ranged from 4,286 bp (V105_16) to 4,517 bp (V105_1), with a mol G + C content ranging from 54.32% (V105_1 and V105_6 T ) to 54.56% (V105_12 and V105_16).The GenBank and SRA accession numbers for the strains are shown in Table 1.
The partial 16S rRNA gene sequences of these isolates exhibited pairwise sequence similarities of 99.39% to 99.45% similarity to other Scandinavium type strains, as determined by the EzBioCloud pipeline (Table 1).Phylogenetic analysis based on 16S rRNA gene sequences grouped these isolates closely with Scandinavium type strains, and were clearly separated from the other type strains within the Enterobacteriaceae family, except Pluralibacter gergoviae, the closest related species to the Scandinavium genus (Suppl. Figure 1).Additionally, RAPD-PCR and rep-PCR fingerprinting of these isolates revealed highly similar fingerprint profiles between V105_1 and V105_6 T , and V105_12 and V105_16 (Suppl.Figure 2).
Based on MLSA phylogenetic analysis, including type strains of Scandinavium species like S. goeteborgense CCUG 66741 T, S. hiltneri H11S7T, S. manionii H17S15T, and S. tedordense TWS1aT, all lettuce isolates were closely related to each other.They showed closer affinity to Scandinavium species type strains than to those of Pluralibacter species.Importantly, they formed a distinct group separate from all Scandinavium species type strains, indicating a novel species (Fig. 1).ANI and dDDH values of all four isolates were below the species delineation thresholds (< 70% for dDDH, < 96% for ANI) when compared to other Scandinavium spp.type strains (Table 2) [29].Additional genome sequences related to our isolates were searched in NCBI database using 6 housekeeping genes (16S rRNA, atpD, dnaJ, infB, gyrB, and rpoB) by BLASTn, and the top-100 related genome sequences were extracted.However, dDDH analysis results indicated that there were no further related whole genome sequences among these strains.The highest ANI value (86.29%) was between strain V105_12 and S. goeteborgense CCUG 66741 T , and the lowest (85.47%) was between strain V105_16 and S. tedordense TWS1a T (Table 2).Notably, all four isolates showed the same highest dDDH value of 31.1% similarity compared to S. goeteborgense CCUG 66741 T , and the lowest value of 29.8% was for V105_12 and V105_16 compared to S. tedordense TWS1a T (Table 2).
Furthermore, a heatmap based on accessory genes (PAN-AROO v. 1.4.2) [30] indicated that V105_1 and V105_6 T , and V105_12 and V105_16 are clonal isolates (Suppl.Figure 3), supporting the RAPD-PCR and rep-PCR fingerprinting results.The whole genome phylogenetic tree clustered the four strains closely with Scandinavium type strains, distinct with type strains of other genera within the family Enterobacteriaceae such as Pluralibacter and Enterobacter (Fig. 2).Altogether, these findings indicated that strain V105_6 T could be regarded as a novel species in the Scandinavium genus.

Antibiotic Resistance
The antibiotic resistance profile of the isolates was determined by the disc diffusion method following CLSI guidelines [23].The results showed that all four strains were resistant to ampicillin, while all strains except V105_1 were resistant also to cefoxitin.Strain V105_1 exhibited an intermediate response to this antibiotic.Moreover, an intermediate resistance occurred for both strains V105_6 T and V105_12, while strain V105_1 showed an intermediate response to cefotaxime (Suppl.Table 1).According to the BLASTp results, the QnrB family protein sequences of all four isolates were identical.When compared to the novel quinolone resistance pentapeptide repeat protein QnrB96, they showed 95.79% sequence identity with 9 aa (amino acid) substitutions, while QnrB1 exhibited 86.92% sequence identity with 28 aa substitutions (Suppl.Figure 4).This indicates that the four isolates share significant similarity with QnrB96, which was identified in S. goeteborgense CCUG 66741 T .

Fatty Acid Methyl Ester Analysis
The principal fatty acids identified included C 12:0 , C 14:0 , C 16:0 , C 18:1 ω7c, summed feature 2 (C 14:0 3-OH and/or iso-C 16:1 ) and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c).All cellular fatty acid profiles for Scandinavium species type strains and potential novel species candidates were indicated in Table 4.The fatty acid compositions of strain V105_6 T and V105_16 exhibited remarkable similarity with strains of previously examined species of Scandinavium.

Virulence Genes
Our isolates were found to possess 34-43 virulence genes according to the VFDB, including flagella structure, capsule, and a T6SS.The presence of the T6SS genes was further identified with 101-103 different genes against the SecReT6

Conclusion
The results of phenotypic, biochemical analysis, phylogenetic and genomic analyses indicate that strain V105_6 T (= LMG 33389 T = DSM 117134 T ) represents a novel species within the genus Scandinavium, for which the name Scandinavium lactucae sp.nov. is proposed.

Emendation of Genus Scandinavium
The Genus Scandinavium is as described by Marathe et al.
[2] and the description is based on the data from [2,4] and this study.Some species in the genus may be ornithine decarboxylase, β-glucuronidase, and α-glucosidase positive.
The genus Scandinavium has been reported in a wide variety of habitats, and strains have been isolated from human wound infection, rhizosphere soil, bleeding lesions of broadleaf hosts, and lettuce.The major fatty acids are C 16:0 , C 18:1 ω7c, C 17:0 cyclo, and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c).The type species of the genus is Scandinavium goeteborgense (CCUG 66741 T = CECT 9823 T = NCTC 14286 T ).
The type strain is V105_6 T (= LMG 33389 T = DSM 117134 T ).The GenBank accession number of the whole genome sequence of the type strain V105_6 T is JAWXRD000000000.
The accession numbers at DDBJ/ENA/GenBank for the draft genomes are listed in Table 1.

Fig. 2
Fig.2Phylogenetic tree based on whole genome sequences of strains V105_1, V105_6 T , V105_12, and V105_16 with closely related Scandinavium type strains including some Enterobacteriaceae type strains.The tree was constructed by extracting and concatenating 100 conserved coding sequences using BV-BRC server[11].The MAFFT

Table 1
Summary of whole genome sequencing results of Scandinavium isolates from lettuce

Table 3
[6]notypic profiles of all known members of the genus Scandinavium.Other type strains data were adapted from previous study[6]